The present invention, in some embodiments thereof, relates to microbiology and, more particularly, but not exclusively, to bacterial sample concentration and depletion techniques.
Microbial contamination of potable water can put water consumers at risk even at low concentrations and short exposure time periods, and therefore rapid, accurate and sensitive pathogen detection techniques are in ever-present demand. Similarly, rapid detection and unambiguous identification of pathogens is crucial in life-threatening medical situations. A key step in most contamination detection methods for pathogenic microorganisms in liquid media is concentration, which is necessary due to the typically low ambient densities of pathogens in the media which are usually lower than the limit of detection.
The emergence of new rapid detection and analysis of microorganisms is closely linked to the development of new concentration methods. For example, immunological or PCR based methods are used after an incubation step because of a minimal detection limit, which hampers the process of rapid detection. In research there has been an emphasis on the development of high throughput screening (HTS) for microbial cell-identification assays using, for example, real-time PCR, microarrays, immunofluorescent methods and combinations thereof. These research studies require a process to lyse bacterial cells, purify and label nucleic acids, and detect organism signatures using microarrays and other HTS techniques. Also, work has been conducted to automate separation of DNA and whole cells from soils, sediments, food, and water. However, these rapid procedures require extensive and time-consuming cell-sample preparations since a high throughput assay usually only reflects the endpoint detector step and not the entire process of sample concentration and preparation which is needed for optimal detection.
One of the most widely used techniques for concentration of microorganisms in liquid media utilizes a size-cutoff membrane filters (MF) through which the liquid media is filtered and the pathogen's cells are stopped by the membrane and thus concentrated. Such a technique is usually the method of choice for the determination of total coliforms, a commonly used indicator of fecal contamination in water. While the MF method is simple and yields definitive results, factors such as elevated turbidity, ambient particles and sediments, resulting in membrane filter blockage, and other filter-type related factors such as inhibition of microbial growth at grid lines, abnormal spreading of colonies, non wetting areas, brittleness, severe wrinkling and decreased recovery, may severely influence the viability, accuracy and sensitivity of the procedure and lead to false and ill-reproducible results. Moreover, the need to concentrate large volumes of the tested media in order to compensate for spatial and temporal variations in pathogen occurrence, increases the probability of membrane filter blockage.
Alternatively, microorganisms can be concentrated and separated from their constituent matrix components in a number of ways. For example, whole bacterial cells have been isolated from food using reagents such as hydroxyapatite. Antibodies coupled to magnetic beads were used to separate specific organisms from human fluids, food, and water, and are widely used in many different applications. Novel methods for semi-specific capture of microorganisms using cell-surface derived lectins and carbohydrates have also been proposed.
Peptides and peptide-mimetic compounds have been investigated for use for broad-spectrum and/or specific binding of microorganisms. Specific capture of mycobacterium in milk was attempted using peptide conjugation to a polymer [1]. Furthermore, antimicrobial peptides linked to surfaces were used for killing [2], immobilization [3] and detection [4] of bacteria.
WO2006/035431 teaches a novel class of antimicrobial polymeric agents which are designed to specifically bind and kill the cell via a two-step mechanism involving an initial high affinity interaction with the microbial external membranes followed by internalization which eventually kills the cell. WO2006/035431 further discloses pharmaceutical compositions and food additives containing these agents as well as methods of treating medical conditions associated with pathological microorganisms. Hence, all the applications described by WO2006/035431 are related to cell killing.